A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Predicted coronavirus Nsp5 protease cleavage sites in the human proteome.

BACKGROUND: The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. RESULTS: Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. CONCLUSIONS: This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of coronavirus infection.

A Novel Proteomic Method Reveals NLS Tagging of T-DM1 Contravenes Classical Nuclear Transport in a Model of HER2-Positive Breast Cancer.

The next breakthrough for protein therapeutics is effective intracellular delivery and accumulation within target cells. Nuclear localization signal (NLS)-tagged therapeutics have been hindered by the lack of efficient nuclear localization due to endosome entrapment. Although development of strategies for tagging therapeutics with technologies capable of increased membrane penetration has resulted in proportional increased potency, nonspecific membrane penetration limits target specificity and, hence, widespread clinical success. There is a long-standing idea that nuclear localization of NLS-tagged agents occurs exclusively via classical nuclear transport. In the present study, we modified the antibody-drug conjugate trastuzumab-emtansine (T-DM1) with a classical NLS linked to cholic acid (cell accumulator [Accum]) that enables modified antibodies to escape endosome entrapment and increase nuclear localization efficiency without abrogating receptor targeting. In parallel, we developed a proteomics-based method to evaluate nuclear transport. Accum-modified T-DM1 significantly enhanced cytotoxic efficacy in the human epidermal growth factor receptor 2 (HER2)-positive SKBR3 breast cancer system. We discovered that efficacy was dependent on the nonclassical importin-7. Our evaluation reveals that when multiple classical NLS tagging occurs, cationic charge build-up as opposed to sequence dominates and becomes a substrate for importin-7. This study results in an effective target cell-specific NLS therapeutic and a general approach to guide future NLS-based development initiatives.

Targeted and Untargeted Proteomics Approaches in Biomarker Development.

An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for new and better biomarkers. Here, what leads to successful biomarker development is reviewed and how these features may be applied in the context of proteomic biomarker research is considered. The "fit-for-purpose" approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using MS-based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. i) The reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how MS can improve, complement, or replace existing clinically important assays in the future are discussed.

Dehydroascorbic acid S-Thiolation of peptides and proteins: Role of homocysteine and glutathione.

Ascorbic acid (vitamin C) plays a significant role in the prevention of oxidative stress. In this process, ascorbate is oxidized to dehydroascorbate (DHA). We have investigated the impact of DHA on peptide/protein intramolecular disulfide formation as well as S-glutathionylation and S-homocysteinylation. S-glutathionylation of peptides/proteins is a reversible, potential regulatory mechanism in oxidative stress. Although the exact role of protein S-homocysteinylation is unknown, it has been proposed to be of importance in pathobiological processes such as onset of cardiovascular disease. Using an in vitro model system, we demonstrate that DHA causes disulfide bond formation within the active site of recombinant human glutaredoxin (Grx-1). DHA also facilities the formation of S-glutathionylation and S-homocysteinylation of a model peptide (AcFHACAAK) as well as Grx-1. We discuss the possible mechanisms of peptide/protein S-thiolation, which can occur either via thiol exchange or a thiohemiketal intermediate. A thiohemiketal DHA-peptide adduct was detected by mass spectrometry and its location on the peptide/protein cysteinyl thiol group was unambiguously confirmed by tandem mass spectrometry. This demonstrates that peptide/protein S-thiolation mediated by DHA is not limited to thiol exchange reactions but also takes place directly via the formation of a thiohemiketal peptide intermediate. Finally, we investigated a potential reducing role of glutathione (GSH) in the presence of S-homocysteinylated peptide/protein adducts. S-homocysteinylated AcFHACAAK, human hemoglobin α-chain and Grx-1 were incubated with GSH. Both peptide and proteins were reduced, and homocysteine replaced with GS-adducts by thiol exchange, as a function of time.

The response to neoadjuvant chemoradiotherapy with 5-fluorouracil in locally advanced rectal cancer patients: a predictive proteomic signature.

BACKGROUND: Colorectal cancer is the third most common and the fourth most lethal cancer in the world. In the majority of cases, patients are diagnosed at an advanced stage or even metastatic, thus explaining the high mortality. The standard treatment for patients with locally advanced non-metastatic rectal cancer is neoadjuvant radio-chemotherapy (NRCT) with 5-fluorouracil (5-FU) followed by surgery, but the resistance rate to this treatment remains high with approximately 30% of non-responders. The lack of evidence available in clinical practice to predict NRCT resistance to 5-FU and to guide clinical practice therefore encourages the search for biomarkers of this resistance. METHODS: From twenty-three formalin-fixed paraffin-embedded (FFPE) biopsies performed before NRCT with 5-FU of locally advanced non-metastatic rectal cancer patients, we extracted and analysed the tumor proteome of these patients. From clinical data, we were able to classify the twenty-three patients in our cohort into three treatment response groups: non-responders (NR), partial responders (PR) and total responders (TR), and to compare the proteomes of these different groups. RESULTS: We have highlighted 384 differentially abundant proteins between NR and PR, 248 between NR and TR and 417 between PR and TR. Among these proteins, we have identified many differentially abundant proteins identified as having a role in cancer (IFIT1, FASTKD2, PIP4K2B, ARID1B, SLC25A33: overexpressed in TR; CALD1, CPA3, B3GALT5, CD177, RIPK1: overexpressed in NR). We have also identified that DPYD, the main degradation enzyme of 5-FU, was overexpressed in NR, as well as several ribosomal and mitochondrial proteins also overexpressed in NR. Data are available via ProteomeXchange with identifier PXD008440. CONCLUSIONS: From these retrospective study, we implemented a protein extraction protocol from FFPE biopsy to highlight protein differences between different response groups to RCTN with 5-FU in patients with locally advanced non-metastatic rectal cancer. These results will pave the way for a larger cohort for better sensitivity and specificity of the signature to guide decisions in the choice of treatment.